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Chondrosarcoma (CS) is a rare malignant bone sarcoma that primarily affects cartilage cells in the femur and pelvis. While most subtypes exhibit slow growth with a very good prognosis, some aggressive subtypes have a poorer overall survival. CS is known for its resistance to chemotherapy and radiotherapy, leaving surgery as the sole effective therapeutic option. Cold physical plasma (CPP) has been explored in vitro as a potential therapy, demonstrating positive anti-tumor effects on CS cells. This study investigated the synergistic effects of combining CPP with cytostatics on CS cells. The chemotherapeutic agents cisplatin, doxorubicin, and vincristine were applied to two CS cell lines (CAL-78 and SW1353). After determining their IC20 and IC50, they were combined with CPP in both cell lines to assess their impact on the cell proliferation, viability, metabolism, and apoptosis. This combined approach significantly reduced the cell proliferation and viability while increasing the apoptosis signals compared to cytostatic therapy alone. The combination of CPP and chemotherapeutic drugs shows promise in targeting chemoresistant CS cells, potentially improving the prognosis for patients in clinical settings.
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Apoptose , Neoplasias Ósseas , Proliferação de Células , Sobrevivência Celular , Condrossarcoma , Doxorrubicina , Gases em Plasma , Condrossarcoma/tratamento farmacológico , Condrossarcoma/patologia , Humanos , Gases em Plasma/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Neoplasias Ósseas/terapia , Antineoplásicos/farmacologia , Cisplatino/farmacologia , Vincristina/farmacologia , Terapia CombinadaRESUMO
Although Ewing's sarcoma (ES) is a rare, but very aggressive tumor disease affecting the musculoskeletal system, especially in children, it is very aggressive and difficult to treat. Although medical advances and the establishment of chemotherapy represent a turning point in the treatment of ES, resistance to chemotherapy, and its side effects, continue to be problems. New treatment methods such as the application of cold physical plasma (CPP) are considered potential supporting tools since CPP is an exogenous source of reactive oxygen and nitrogen species, which have similar mechanisms of action in the tumor cells as chemotherapy. This study aims to investigate the synergistic effects of CPP and commonly used cytostatic chemotherapeutics on ES cells. The chemotherapy drugs doxorubicin and vincristine, the most commonly used in the treatment of ES, were applied to two different ES cell lines (RD-ES and A673) and their IC20 and IC50 were determined. In addition, individual chemotherapeutics in combination with CPP were applied to the ES cells and the effects on cell growth, cell viability, and apoptosis processes were examined. A single CPP treatment resulted in the dose-dependent growth inhibition of ES cells. The combination of different cytostatics and CPP led to significant growth inhibition, a reduction in cell viability, and higher rates of apoptosis compared to cells not additionally exposed to CPP. The combination of CPP treatment and the application of cytostatic drugs to ES cells showed promising results, significantly enhancing the cytotoxic effects of chemotherapeutic agents. These preclinical in vitro data indicate that the use of CPP can enhance the efficacy of common cytostatic chemotherapeutics, and thus support the translation of CPP as an anti-tumor therapy in clinical routine.
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Antineoplásicos , Neoplasias Ósseas , Citostáticos , Sarcoma de Ewing , Criança , Humanos , Sarcoma de Ewing/patologia , Citostáticos/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Vincristina/farmacologia , Vincristina/uso terapêutico , Doxorrubicina/uso terapêutico , Neoplasias Ósseas/metabolismo , Linhagem Celular TumoralRESUMO
Chondrosarcoma is the second most common malign bone tumor in adults. Surgical resection of the tumor is recommended because of its resistance to clinical treatment such as chemotherapy and radiation therapy. Thus, the prognosis for patients mainly depends on sufficient surgical resection. Due to this, research on alternative therapies is needed. Cold atmospheric plasma (CAP) is an ionized gas that contains various reactive species. Previous studies have shown an anti-oncogenic potential of CAP on different cancer cell types. The current study examined the effects of treatment with CAP on two chondrosarcoma cell lines (CAL-78, SW1353). Through proliferation assay, the cell growth after CAP-treatment was determined. A strong antiproliferative effect for both cell lines was detected. By fluorescein diacetate (FDA) assay and ATP release assay, alterations in the cell membrane and associated translocation of low molecular weight particles through the cytoplasmic membrane were observed. In supernatant, the non-membrane-permeable FDA and endogenously synthesized ATP detected suggest an increased membrane permeability after CAP treatment. Similar results were shown by the dextran-uptake assay. Furthermore, fluorescence microscopic G-/F-actin assay was performed. G- and F-actin were selectively dyed, and the ratio was measured. The presented results indicate CAP-induced changes in cell membrane function and possible alterations in actin-cytoskeleton, which may contribute to the antiproliferative effects of CAP.
Assuntos
Permeabilidade da Membrana Celular , Membrana Celular/efeitos dos fármacos , Proliferação de Células , Condrossarcoma/metabolismo , Gases em Plasma/farmacologia , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , HumanosRESUMO
BACKGROUND: Cold atmospheric plasma (CAP) is increasingly used in the field of oncology. Many of the mechanisms of action of CAP, such as inhibiting proliferation, DNA breakage, or the destruction of cell membrane integrity, have been investigated in many different types of tumors. In this regard, data are available from both in vivo and in vitro studies. Not only the direct treatment of a tumor but also the influence on its blood supply play a decisive role in the success of the therapy and the patient's further prognosis. Whether the CAP influences this process is unknown, and the first indications in this regard are addressed in this study. METHODS: Two different devices, kINPen and MiniJet, were used as CAP sources. Human endothelial cell line HDMEC were treated directly and indirectly with CAP, and growth kinetics were performed. To indicate apoptotic processes, caspase-3/7 assay and TUNEL assay were used. The influence of CAP on cellular metabolism was examined using the MTT and glucose assay. After CAP exposure, tube formation assay was performed to examine the capillary tube formation abilities of HDMEC and their migration was messured in separate assays. To investigate in a possible mutagenic effect of CAP treatment, a hypoxanthine-guanine-phosphoribosyl-transferase assay with non malignant cell (CCL-93) line was performed. RESULTS: The direct CAP treatment of the HDMEC showed a robust growth-inhibiting effect, but the indirect one did not. The MMT assay showed an apparent reduction in cell metabolism in the first 24 h after CAP treatment, which appeared to normalize 48 h and 72 h after CAP application. These results were also confirmed by the glucose assay. The caspase 3/7 assay and TUNEL assay showed a significant increase in apoptotic processes in the HDMEC after CAP treatment. These results were independent of the CAP device. Both the migration and tube formation of HDMEC were significant inhibited after CAP-treatment. No malignant effects could be demonstrated by the CAP treatment on a non-malignant cell line.
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Inibidores da Angiogênese/farmacologia , Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Gases em Plasma/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismoRESUMO
Osteosarcoma and Ewing's sarcoma are the most common malignant bone tumors. Conventional therapies such as polychemotherapy, local surgery, and radiotherapy improve the clinical outcome for patients. However, they are accompanied by acute and chronic side effects that affect the quality of life of patients, motivating novel research lines on therapeutic options for the treatment of sarcomas. Previous experimental work with physical plasma operated at body temperature (cold atmospheric plasma, CAP) demonstrated anti-oncogenic effects on different cancer cell types. This study investigated the anti-cancer effect of CAP on two bone sarcoma entities, osteosarcoma and Ewing's sarcoma, which were represented by four cell lines (U2-OS, MNNG/HOS, A673, and RD-ES). A time-dependent anti-proliferative effect of CAP on all cell lines was observed. CAP-induced alterations in cell membrane functionality were detected by performing a fluorescein diacetate (FDA) release assay and an ATP release assay. Additionally, modifications of the cell membrane and modifications in the actin cytoskeleton composition were examined using fluorescence microscopy monitoring dextran-uptake assay and G-/F-actin distribution. Furthermore, the CAP-induced induction of apoptosis was determined by TUNEL and active caspases assays. The observations suggest that a single CAP treatment of bone sarcoma cells may have significant anti-oncogenic effects and thus may be a promising extension to existing applications.
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Neoplasias Ósseas/tratamento farmacológico , Osteossarcoma/tratamento farmacológico , Gases em Plasma/farmacologia , Sarcoma de Ewing/tratamento farmacológico , Apoptose , Neoplasias Ósseas/patologia , Proliferação de Células , Humanos , Osteossarcoma/patologia , Sarcoma de Ewing/patologia , Células Tumorais CultivadasRESUMO
BACKGROUND/AIM: Cold physical plasma (CPP) has emerged as an effective therapy in oncology by inducing cytotoxic effects in various cancer cells, including chondrosarcoma (CS), Ewing's sarcoma (ES), and osteosarcoma (OS). The current study investigated the impact of CPP on cell motility in CS (CAL-78), ES (A673), and OS (U2-OS) cell lines, focusing on the actin cytoskeleton. MATERIALS AND METHODS: The CASY Cell Counter and Analyzer was used to study cell proliferation and determine the optimal concentrations of fetal calf serum to maintain viability without stimulation of cell proliferation. CellTiter-BlueCell viability assay was used to determine the effects of CPP on the viability of bone sarcoma cells. The Radius assay was used to determine cell migration. Staining for Deoxyribonuclease I, G-actin, and F-actin was used to assay for the effects on the cytoskeleton. RESULTS: Reductions in cell viability and motility were observed across all cell lines following CPP treatment. CPP induced changes in the actin cytoskeleton, leading to decreased cell motility. CONCLUSION: CPP effectively reduces the motility of bone sarcoma cells by altering the actin cytoskeleton. These findings underscore CPP's potential as a therapeutic tool for bone sarcomas and highlight the need for further research in this area.
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Citoesqueleto de Actina , Neoplasias Ósseas , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Citoesqueleto , Gases em Plasma , Humanos , Movimento Celular/efeitos dos fármacos , Gases em Plasma/farmacologia , Linhagem Celular Tumoral , Neoplasias Ósseas/patologia , Neoplasias Ósseas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Osteossarcoma/patologia , Osteossarcoma/metabolismo , Actinas/metabolismo , Sarcoma/patologia , Sarcoma/metabolismoRESUMO
Ewing's sarcoma (ES) is the second most common bone tumor in children and adolescents and is highly malignant. Although the new chemotherapy has significantly improved the survival rate for ES from about 10 to 75%, the survival rate for metastatic tumors remains around 30%. This treatment is often associated with various side effects that contribute to the suffering of the patients. Cold physical plasma (CPP), whether used alone or in combination with current chemotherapy, is considered a promising adjunctive tool in cancer treatment. This study aims to investigate the synergistic effects of CPP in combination with cytostatic chemotherapeutic agents that are not part of current ES therapy. Two different ES cell lines, RD-ES and A673, were treated with the determined IC20 concentrations of the chemotherapeutic agents cisplatin and methotrexate (MTX) in combination with CPP. The effects on population doubling, cell viability, and apoptotic processes within these cell lines were assessed. This combination therapy has led to a reduction of population doubling and cell viability, as well as an increase in apoptotic activity in cells compared to CPP monotherapy. The results of this study provide evidence that combining CPP with non-common chemotherapy drugs such as MTX and CIS in the treatment of ES enhances the anticancer effects of these drugs. These findings open up new possibilities for the effective use of these drugs against ES.
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Neoplasias Ósseas , Sarcoma de Ewing , Criança , Adolescente , Humanos , Sarcoma de Ewing/patologia , Neoplasias Ósseas/patologia , Terapia Combinada , Apoptose , Cisplatino/farmacologia , Cisplatino/uso terapêuticoRESUMO
Judo is an Olympic sport, and the way of its performing can lead to repetitive blunt injuries on head and ears. The chronic consequences of such traumata on the auricle are the formation of so-called cauliflower ear. This condition is painful, can lead to interruptions in the training process and long-term consequences for the athlete's health. There is limited knowledge of epidemiological data about cauliflower ear deformities in judo. Evaluation of the prevalence of cauliflower ear among judokas based on their profile pictures on the international judo federation was performed. A large cohort of judo athletes from around the world was studied. Two different classifications for the severity of ear deformities were used. Statistical calculations of the collected data and correlations to different parameters were performed. Images of 1632 top athletes were evaluated in the study. Ear deformities were found in 55.5% of the judokas. There was gender-specific differences. Male athletes were affected much more often than female athletes. In addition, ear deformities were more pronounced in male athletes. A correlation was found between the age of the athletes and the presence of an ear deformity. It has also been shown that judokas with a high world ranking are more likely to have an ear deformity. Ear deformities are a common consequence of injury among leading judo athletes. The current study represents the largest and high heterogeny cohort ever conducted on the prevalence of cauliflower ear in judoka. Knowledge of the prevalence of cauliflower ear in judoka based on reliable data from this study, may be important prerequisites for further studies on the impact of this traumatic consequence on training preparation and judoka health.
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Artes Marciais , Humanos , Masculino , Feminino , Prevalência , Artes Marciais/lesões , Orelha Externa , Fatores Sexuais , AtletasRESUMO
Renal cell carcinoma (RCC) is the third most common urological tumor and has an extremely poor prognosis after metastasis has occurred. Therapeutic options are highly restricted, primarily due to resistance to classical chemotherapeutics. The development of new, innovative therapeutic procedures is thus of great urgency. In the present study, the influence of non-invasive physical plasma (NIPP) on malignant and non-malignant renal cells is characterized. The biological efficacy of NIPP has been demonstrated in malignant renal cell lines (786-O, Caki-1) and non-malignant primary human renal epithelial cells (HREpC). The cell responses that were experimentally examined were cell growth (cell number determination, calculation of growth rate and doubling time), cell motility (scratch assay, invasiveness assay), membrane integrity (uptake of fluorescent dye, ATP release), and induction of apoptosis (TUNEL assay, caspase-3/7 assay, comet assay). A single NIPP treatment of the malignant cells significantly inhibited cell proliferation, invasiveness, and metastasis. This treatment has been attributed to the disruption of membrane functionality and the induction of apoptotic mechanisms. Comparison of NIPP sensitivity of malignant 786-O and Caki-1 cells with non-malignant HREpC cells showed significant differences. Our results suggest that renal cancer cells are significantly more sensitive to NIPP than non-malignant renal cells. Treatment with NIPP could represent a promising innovative option for the therapy of RCC and might supplement established treatment procedures. Of high clinical relevance would be the chemo-sensitizing properties of NIPP, which could potentially allow a combination of NIPP treatment with low-dose chemotherapy.
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BACKGROUND: The use of cold atmospheric plasma (CAP) in oncology has been intensively investigated over the past 15 years as it inhibits the growth of many tumor cells. It is known that reactive oxidative species (ROS) produced in CAP are responsible for this effect. However, to translate the use of CAP into medical practice, it is essential to know how CAP treatment affects non-malignant cells. Thus, the current in vitro study deals with the effect of CAP on human bone cancer cells and human osteoblasts. Here, identical CAP treatment regimens were applied to the malignant and non-malignant bone cells and their impact was compared. METHODS: Two different human bone cancer cell types, U2-OS (osteosarcoma) and A673 (Ewing's sarcoma), and non-malignant primary osteoblasts (HOB) were used. The CAP treatment was performed with the clinically approved kINPen MED. After CAP treatment, growth kinetics and a viability assay were performed. For detecting apoptosis, a caspase-3/7 assay and a TUNEL assay were used. Accumulated ROS was measured in cell culture medium and intracellular. To investigate the influence of CAP on cell motility, a scratch assay was carried out. RESULTS: The CAP treatment showed strong inhibition of cell growth and viability in bone cancer cells. Apoptotic processes were enhanced in the malignant cells. Osteoblasts showed a higher potential for ROS resistance in comparison to malignant cells. There was no difference in cell motility between benign and malignant cells following CAP treatment. CONCLUSIONS: Osteoblasts show better tolerance to CAP treatment, indicated by less affected viability compared to CAP-treated bone cancer cells. This points toward the selective effect of CAP on sarcoma cells and represents a further step toward the clinical application of CAP.
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(1) Background: Chondrosarcoma (CS) is a malignant primary bone tumor with a cartilaginous origin. Its slow cell division and severely restricted vascularization are responsible for its poor responsiveness to chemotherapy and radiotherapy. The decisive factor for the prognosis of CS patients is the only adequate therapy-surgical resection. Cold atmospheric pressure plasma (CAP) is emerging as a new option in anti-cancer therapy. Its effect on chondrosarcomas has been poorly investigated. (2) Methods: Two CS cell lines-SW 1353 and CAL 78-were used. Various assays, such as cell growth kinetics, glucose uptake, and metabolic activity assay, along with two different apoptosis assays were performed after CAP treatment. A radius cell migration assay was used to examine cell motility. (3) Results: Both cell lines showed different growth behavior, which was taken into account when using the assays. After CAP treatment, a reduction in metabolic activity was observed in both cell lines. The immediate effect of CAP showed a reduction in cell numbers and in influence on this cell line's growth rate. The measurement of the glucose concentration in the cell culture medium showed an increase after CAP treatment. Live-dead cell imaging shows an increase in the proportion of dead cells over the incubation time for both cell lines. There was a significant increase in apoptotic signals after 48 h and 72 h for both cell lines in both assays. The migration assay showed that CAP treatment inhibited the motility of chondrosarcoma cells. The effects in all experiments were related to the duration of CAP exposure. (4) Conclusions: The CAP treatment of CS cells inhibits their growth, motility, and metabolism by initiating apoptotic processes.
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BACKGROUND/AIM: The typical insulin deficiency in type 1 diabetes mellitus has general effects on metabolism and also affects bone quality. MATERIALS AND METHODS: Two diabetic rat lines (BB/OK; BB.6KWR) and two non-diabetic rat strains (KWR and BB.14+18KWR), as control group, were included in the study. Bone mineral density, bone mineral content and body structure measurements were performed. The measurements took place before the onset of diabetes mellitus Results: A comparison of the groups showed increased bone density values of the diabetic rats in relation to the control groups. A new finding of increased bone density in the diabetic rats occurs. CONCLUSION: Diabetic rats showed no osteoporotic bone metabolism before the onset of clinically relevant type 1 diabetes mellitus, but rather increased bone metabolic activity.
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Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Animais , Densidade Óssea , Osso e Ossos/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Insulina , RatosRESUMO
BACKGROUND/AIM: Mammary carcinoma (MC) remains one of the leading causes of morbidity and mortality in the female population worldwide. Cold physical plasma at atmospheric pressure (CAP) has an antioncogenic effect on tumor cells, and its anticancer properties may complement or even extend existing treatment options. In the present study, the efficacy of CAP was characterized on an MC in vitro cell culture system. MATERIALS AND METHODS: MC cells (MCF-7, MDA-MB-231) were directly treated with CAP or incubated with CAP-treated cell culture medium. Cell growth, cell mobility and apoptosis were subsequently analyzed. RESULTS: A single treatment of MC cells with CAP and CAP treated medium led to a treatment-time dependent reduction of cell growth. Furthermore, CAP exposure led to a loss of cellular motility and induced apoptosis. CONCLUSION: Due to its anticancer properties, CAP treatment is an innovative and promising physical approach to expand and complement the treatment options for MC. In particular, a combination of CAP application with surgical and/or chemotherapeutic interventions might significantly improve the therapeutic outcomes.
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Argônio/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Gases em Plasma/uso terapêutico , Argônio/farmacologia , Pressão Atmosférica , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Cinética , Gases em Plasma/farmacologiaRESUMO
BACKGROUND: Physical plasma is a mixture of reactive particles and electromagnetic radiation. Due to the antimicrobial, immunomodulatory, anti-inflammatory, wound-healing promoting, and antineoplastic effects of body tempered physical plasma under atmospheric pressure (cold atmospheric plasma: CAP), CAP therapy is increasingly becoming the focus of surgical and oncological disciplines. However, when applied in practice, a potential emission of harmful noxae such as toxic nitrogen oxides must be taken into account, which was investigated in the following study. MATERIALS AND METHODS: MiniJet-R Ar CAP device was characterized with respect to NOX-specific spectra, ultraviolet radiation C (UVC) intensity in the range of 200-275 nm and the formation of NOX gases. Instrument-specific parameters such as gas flow, energy setting of the high-frequency generator, and flow rate of the carrier gas Ar were varied. To test the toxic properties of the NO2 concentrations formed by CAP, SK-OV-3 human ovarian cancer cells were incubated with different NO2 concentrations and cell growth was monitored for 120 h. RESULTS: The operation of MiniJet-R led to the formation of NO2 in the proximity of the CAP effluent. Synthesis of NO led to a NO-specific spectrum in the range of 100-275 nm, whereby UVC radiation produced reached intensities of up to 90 mW/m2 NO gas itself, however, was not detectable, as it was converted to NO2 rapidly. Cell culture incubation experiments demonstrated that NO2 in these concentration ranges had no influence on the cell growth of human cancer cells. CONCLUSION: Although no limit values were exceeded in the present study, the emission of high-energy UVC radiation and toxic NO2 is a risk factor with regard to the legal regulations on workplace protection (operator hazard) and the approval of medical devices (patient hazard). This is important for considerations regarding treatment frequency and duration. The growth inhibitory effect of CAP treatment on human cancer cells principally suggests a medical application of the MiniJet-R device, although more extensive studies will have to follow.
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Pressão Atmosférica , Dióxido de Nitrogênio/toxicidade , Gases em Plasma/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Óxido Nítrico/análise , Dióxido de Nitrogênio/análise , Raios UltravioletaRESUMO
BACKGROUND: Cold atmospheric plasma (CAP) has a variety of anticancer effects on different cancer cell types. In osteosarcoma (OS) cells, CAP reduces growth and motility, induces apoptosis, and alters secretion of cellular factors. The influence of CAP on membrane integrity of OS cells is unknown. MATERIALS AND METHODS: Two different OS cell lines (U-2 OS and MNNG-HOS) were treated with CAP. Proliferation assays for cell growth after treatment was performed. Alterations in membrane permeability and the associated translocation of low molecular weight particles through the cytoplasmic membrane of OS cells after CAP treatment were shown in fluorescein diacetate (FDA) assays. RESULTS: FDA increasingly passed the membrane after CAP treatment and this effect depended on the duration of treatment. It was also shown that after CAP treatment, FDA was able to diffuse into the cells from the outside as well as out of the cell interior. These effects were observed when CAP-treated buffer was used and therefore no direct contact between cells and CAP occurred. CONCLUSION: The observations suggest that changes in membrane permeability and function may contribute to the antiproliferative effects of CAP.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Osteossarcoma/tratamento farmacológico , Gases em Plasma/uso terapêutico , Linhagem Celular Tumoral , Humanos , Osteossarcoma/patologia , Gases em Plasma/farmacologiaRESUMO
Interaction of Staphylococcus aureus alpha-toxin (hemolysin A, Hla) with eukaryotic cell membranes is mediated by proteinaceous receptors and certain lipid domains in host cell plasma membranes. Hla is secreted as a 33 kDa monomer that forms heptameric transmembrane pores whose action compromises maintenance of cell shape and epithelial tightness. It is not exactly known whether certain membrane lipid domains of host cells facilitate adhesion of Ha monomers, oligomerization, or pore formation. We used sphingomyelinase (hemolysin B, Hlb) expressed by some strains of staphylococci to pre-treat airway epithelial model cells in order to specifically decrease the sphingomyelin (SM) abundance in their plasma membranes. Such a pre-incubation exclusively removed SM from the plasma membrane lipid fraction. It abrogated the formation of heptamers and prevented the formation of functional transmembrane pores. Hla exposure of rHlb pre-treated cells did not result in increases in [Ca2+]i, did not induce any microscopically visible changes in cell shape or formation of paracellular gaps, and did not induce hypo-phosphorylation of the actin depolymerizing factor cofilin as usual. Removal of sphingomyelin from the plasma membranes of human airway epithelial cells completely abrogates the deleterious actions of Staphylococcus aureus alpha-toxin.
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Transportadores de Cassetes de Ligação de ATP/genética , Toxinas Bacterianas/toxicidade , Células Epiteliais/efeitos dos fármacos , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidade , Esfingomielinas/deficiência , Toxinas Bacterianas/genética , Linhagem Celular , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Humanos , Sistema Respiratório/citologiaRESUMO
BACKGROUND/AIM: The structural integrity of the eukaryotic cytoplasmic membrane is of crucial importance for its cell biological function and thus for the survival of the cell. Physical and chemical noxae can interact in various ways with components of the cytoplasmic membrane, influence its permeability and thus mediate toxic effects. In the study presented, changes in membrane permeability were quantified by intracellular accumulation of a fluorescent dye and by the release of the fluorescent dye from dye-loaded cells. MATERIALS AND METHODS: Non-malignant (RC-124) and malignant (786-O, Caki-1) renal cells were permeabilized with different concentrations of Triton X-100. The permeability of the membrane was determined at the single-cell level by the uptake of the dye into the cell inner by flow cytometry. In addition, a fluorescence plate reader was used to detect and quantify the release of the dye into the cell culture supernatant. RESULTS: Both malignant and non-malignant cells showed a dose-dependent alteration of membrane permeability after treatment with Triton X-100. In the presence of the fluorescent dye, significantly more dye was introduced into the permeabilized cells compared to control incubations. Vice versa, Triton X-100-treated and dye-loaded cells released significantly more dye into the cell culture supernatant. CONCLUSION: The combination of measurement of intracellular accumulated and extracellular released dye can quantifiably detect changes in membrane permeability due to cell-membrane damage. The combination of two different measurement methods offers additional value in reliable detection of membrane-damaging, potentially toxic influences.